The focus of research in the Laboratory for Integrative Neuroscience (LIN), Section on Synaptic Pharmacology, is the determination of mechanisms underlying neuromodulation and plasticity and the effects of alcohol and other drugs of abuse on these neuronal functions. As a part of ongoing studies examining alcohol effects on NMDA-type glutamate receptor function, we examined the role of NR2A and NR2B subunits of the receptor in synaptic and non-synaptic receptor function. We also examined long-term synaptic plasticity, in collaboration with the laboratory of Dr. Danny Winder at Vanderbilt University. We inadvertently discovered that the drug NVP-AAM077, touted as a specific blocker of NR2A-containing receptors, was not specific when allowed to equilibrate at receptors prior to agonist application or synaptic activation. This drug appears to act as a slowly-dissociating competitive antagonist at all NMDA receptors. This information allowed us to show that receptors containing the NR2A subunit are not obligatory for long-term synaptic potentiation (LTP), as had been previously suggested. We have also used gene-targeted mice in which the NR2A subunit was knocked out to examine the relative role of this subunit in ethanol inihibition of NMDAR-mediated synaptic transmission. We have observed that ethanol inihibits synaptic transmission in autaptic culture and brain slice preparations from NR2A knockout mice with a potency comparable to that observed in preparations from wildtype mice. We have collaborated with the laboratory of Dr. Kathleen Grant at Wake Forest University to examine behavioral responses to ethanol in NR2A knockout mice using the drug discrimination technique. We have observed that NR2A knockout mice can learn ethanol discrmination, albeit slowly, and that these animals exhibit a loss of generalization to the GABAA receptor-potentiating drug pentobarbital, as well as appearance of generalization to ifenprodil, an antagonist for NR2B-containing NMDA receptors. These findings suggest that the NR2A subunit plays an important role in interactions between glutamatergic and GABAergic transmission during acute intoxication, and that the NR2B subunit can only partially compensate for the loss of NR2A. We have also examined ethanol inhibition of signaling produced by Brain Derived Neurotrophic Factor (BDNF) in granule neurons from the rodent cerebellum. Exposure of these neurons to BDNF activates the multifunctional protein kinase known as Extracellular-signal Regulated Kinase I (ERK1), via increased ERK1 phosphorylation. Ethanol inhibits the BDNF stimulation of phosphorylation at concentrations that are relevant to alcohol intoxication and fetal alcohol effects. This inhibitory effect appears to contribute to some extent to ethanol effects on granule neuron proliferation. However, BDNF is known to have important roles in neuronal differentiation and migration within the cerebellum, and thus we are interested in determining if ethanol affects these aspects of cellular development and if ERK1 is implicated in any actions of ethanol on these processes. This line of research may offer new molecular targets for treatment of fetal alcohol effects. We have continued studies of synaptic plasticity in dorsal striatum, as well as investigations of similar plastic changes in hippocampus and amygdala. In past studies our laboratory and others have shown that endocannabinoids, endogenous lipid metabolites that activate cannabinoid receptors, act as retrograde signals that produce short and long-term changes in neurotransmitter release probability. In particular these molecules are implicated in long-term synaptic depression (LTD), a long-lasting decrease in synaptic efficacy at corticostriatal synapses in dorsal striatum. This form of LTD is known to involve activation of the D2-type dopamine receptor. We have used mice in which the D2 receptor gene promoter drives expression of green fluorescent protein (GFP) in striatal medium spiny neurons that contain D2 receptors, in preference to D1 receptors, to determine if this type of synaptic plasticity depends on the relative abundance of postsynaptic D2 receptors. We find that LTD occurs at excitatory synapses onto all striatal medium spiny neurons regardless of whether they contain primarily D1 or D2 receptors. This finding indicates that postsynaptic D2 receptors may not be necessary for striatal LTD induction. Examination of endocannabinoid-mediated synaptic plasticity in hippocampal slices has revealed that repetitive synaptic activation can produce several forms synaptic plasticity and metaplasticity at inhibitory synapses onto CA1 pyramidal neurons. Activation of synapses onto these neurons at 1 Hz for several minutes produces LTD, similar to that previously observed after stimulation at higher frequencies. Furthermore, the 1 Hz stimulation primes long-term potentiation LTP at excitatory synapses on the same pyramidal neurons. In other words, induction of LTP by 100 Hz stimulation is facilitated following the 1 Hz stimulus train. This is similar to results obtained by Castillo and coworkers with a higher frequency priming stimulus. The most novel form of plasticity/metaplasticity that we have observed following sustained 1 Hz stimulation is a long-lasting increase in endocannabinoid signaling. We have observed a sustained increase in depolarization-induced suppression of inhibition (DSI), a suppression of transmission that is endocannabinoid mediated, following 1 Hz stimulation. Thus, the 1 Hz stimulus primes endocannabinoid production, which in turn primes LTP, and the stimulus also produces LTD of the inhibitory synapses, further priming LTP induction. All of these forms of plasticity and metaplasticity depend on activation of CB1 cannabinoid receptors and mGluR5 metabotropic glutamate receptors. This work reveals a new mechanism through which environmental input to the brain and increased neuronal activity within the brain can bring about long-lasting changes in brain function. Enhanced endocannabinoid function might be involved in addiction as well as learning and memory. Mechanisms involved in endocannabinoid-mediated plasticity and metaplasticity could be targets for new addiction treatments or memory-enhancing agents. We have also continued to explore the molecular mechanisms involved in endocannabinoid production leading to DSI and LTD in amygdala neurons using newly-implemented techniques for isolating neurons with attached GABAergic synaptic boutons. In addition to DSI, we have observed a later-developing component of synaptic depression that requires activation of mGluR5, as we discussed in the annual report for 2004 and in a recently-published paper. This later-developing depression begins ~30 seconds after depolarization and persists for the duration of the recording. The properties of this delayed, mGluR-dependent component of synaptic depression resemble amygdalar LTD. Subsequently, we have observed that a group I mGluR agonist (DHPG) can produce synaptic depression that persists for at least 20 minutes post-drug in this reduced preparation. These findings indicate that LTD can occur under these simple experimental conditions. The use of this preparation should allow us to explore the mechanisms involved in LTD induction and expression in greater detail than would be possible in a brain slice preparation. Endocannabinoid-dependent LTD in the amygdala may play a role in extinction of memories brought about by aversive conditioning. Understanding the molecular basis of this plasticity could lead to better treatments for psychiatric diseases such as posttraumatic stress disorder. These several lines of research should allow us to gain a better understanding of synaptic mechanims of learning and addiction.